!CANCER RESEARCH 56, 2468-2471. June 1. 9961 Advances in Brief Glucose Catabolism in Cancer Cells: Amplification of the Gene Encoding Type II Hexokinase1 Annette Rempel, Saroj P. Mathupala, Constance A. Griffin, Anita L. Hawkins, and Peter L Pedersen2 Departments of Biological Chemistry [A. R.. S. P. M., P. L P.] and Pathology [C. A. G.. A. L H.], The Johns Hopkins University, School of Medicine, Baltimore, Maryland 21205-2185 Abstract Hexokinase type II is highly overexpressed in many cancer cells, where it plays a pivotal role in the high glycolytic phenotype. Here we demon strate by Southern blot analysis and fluorescence in situ hybridization (FISH) that in the rapidly growing rat AS-30D hepatoma cell line, en hanced hexokinase activity is associated with at least a 5-fold amplification of the type II gene relativeto normalhepatocytes.This amplificationis located chromosomally, extends to the whole gene, and most likely occurs at the site of the residentgene. No rearrangementof the gene could be detected. Therefore, overexpression of hexokinase type II in AS-30D hepatoma cells may be based, at least in part, on a stable gene amplifica tion. This is the first report describing the amplification of a hexokinase regulation of the tumor HKII3 gene (4) by elucidating the sequence of its promoter region and identifying activators thereof. The present paper focuses on structural differences between the HKII gene in normal and tumor cells as a possible mechanism for gene induction. For our studies, we used the highly glycolytic, rapidly growing rat hepatoma cell line AS-30D. This cell line has been characterized in detail in this laboratory with respect to its high glycolysis and the role in this process of hexokinase (Refs. 2, 10, and references therein). The data described below indicate that the hexokinase gene is amplified at least 5-fold in AS-30D hepatoma cells relative to normal hepatocytes, and that no rearrangement of the gene occurs. gene in a tumorcell line expressingthe high glycolyticphenotype. Materials Introduction and Methods Cells and Cell Culture. One of the most common and profound phenotypes of malignant tissues, particularly those with the highest growth rates, is their capacity to utilize and catabolize glucose at high rates (1). The high glycolytic rate is important for rapidly proliferating cancers not only as a major energy source, but also to provide such cells with precur sors for nucleotide and lipid biosynthesis. Altered expression of glycolytic enzymes, especially hexokinase (EC 2.7.1.1) is believed to play a major role in this phenomenon (1—3).Hexokinase catalyzes the conversion of glucose to glucose-6-phosphate in the first step of the glycolytic pathway. Hexokinase activity (1—3),mRNA level (3, 4), and transcription rate (5) are increased markedly in rapidly growing tumors. To further potentiate the enhanced hexokinase activity achieved by overexpression, most of the enzyme is bound to the outer mitochondrial membrane, where it has direct access to mitochondri ally generated AlP and is less sensitive to glucose-6-phosphate inhi Clone 9 (CRL 1439), a rat hepatocyte cell line, was obtained from the American Type Culture Collection and grown in RPM! 1640 medium. AS-30D hepatoma cells were grown in the peritoneal cavity of female Sprague-Dawley rats, harvested, and purified as described previously (4). Hepatocytes were isolated from female Sprague-Dawley collagenase perfusion method (12). rats by the Hexokinase Assay. Hexokinase activity was determined spectrophoto metrically on whole-cell lysates using a glucose-6-phosphate dehydrogenase coupled assay (6). Activity is expressed in mUs, I mU defined as the formation of 1 nmol NADPH/min. Southern Blot Analysis. High molecularweight DNA was isolated from AS-30D hepatoma cells and hepatocytes as described (13). DNA (30 p.g) was digested with the indicated restriction enzymes. To avoid technical problems resulting from incomplete hydrolysis, digestions were repeated several times with an excess of restriction enzymes. The digested DNA was fractionated on bition (2, 6). In brain tumors, hexokinase activity is proportional to the a 1% agarose gel and transferred to nylon membranes (Amersham). Probe labeling, hybridization, and detection were performed with the Fluorescein Gene Images System (Amersham) according to the manufacturer's instruc degree of malignancy (7). In addition, Fanciulli et a!. (8) demonstrated that increased hexokinase activity may not only be the consequence of tions. Either the full-length cDNA or a 260-bp fragment corresponding to the position —197 to +63 of rat skeletal muscle HKII (11) were used as probes. altered metabolic requirements of cancer cells but may also be a FISH. The pUC18 plasmid containingthe 3.6-kb cDNA clone of the rat HKII gene was nick translated with biotin-14 dATP (Bethesda Research Laboratories, Gaithersburg, MD), with 25% incorporation as determined by modification per se to increase mitotic activity. Therefore, elucidation of the molecular basis underlying hexokinase overexpression will provide information that is not only useful in explaining the mecha nism of the high glycolytic phenotype but may lead also to new approaches in cancer diagnosis and therapy. Of the four known hexokinase isozymes (I, II, III, and IV), it is the type H, and to a lesser extent the type I isozymes that are overexpressed in rapidly growing, highly glycolytic tumors examined to date (3, 4, 9—11). In a recent study, we addressed for the first time the issue of transcriptional tritium tracer incorporation. Slides with chromosome spreads were made from AS-30D hepatoma cells and clone 9 (normal control), harvested by standard cytogenetic techniques. FISH was performed cations. Probe mix [2X SSCP (1 X SSCP = citrate, 0.02 M sodium phosphate, pH 6.0), sulfate, 5 nW@l biotinylated probe, and 20 as described (14) with modifi 0. 15 M NaCl, 0.015 M sodium 50% formamide, 10% dextran @g4dsalmon sperm DNAI was denatured at 70°Cfor 5 mm, chilled quickly on ice, placed on slides, and hybridized at 37°Covernight. Slides were washed in 50% formamide and 2X SSC(1X SSC = 0.15MNaC1,0.015Msodiumcitrate,pH 7.0)at 43°C for 20 Received 3/1 2/96; accepted 4/23/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with mm, and two changes of 2X SSC at 37°Cfor 5 mm each. Biotinylated probe 18 U.S.C. Section 1734 solely to indicate this fact. using reagents I Supported in part by NIH Grant CA 32742 (to P. L. P.) and NIH Grant 2P30- CA06972 (to C. A. G.). A. R. was an awardee of the Deutsche Forschungsgemeinschaft. 2 To whom correspondence should be addressed, at Department of Biological was detected with FITC-avidin with biotinylated Kit (Oncor antiavidin, Inc., Gaithersburg, MD), following manufacturer's instructions. Chem istry, The Johns Hopkins University, School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185.Phone:(410)955-3827;Fax:(410)955-1944. and amplified from an In Situ Hybridization 3 The abbreviations used are: HKII, hexokinase type II; FISH, fluorescence hybridization; mU, milliunit. 2468 Downloaded from cancerres.aacrjournals.org on May 1, 2016. © 1996 American Association for Cancer Research. in situ GENE AMPLIFICATION OF TYPE ll HEXOK!NASE IN TUMOR CELLS from different Southern blots confirmed the data obtained in the Results and Discussion dilution experiment, and a factor of approximately 5 was calculated for the amplification. Additional support for the ilK!! gene amplifi cation in AS-30D hepatoma cells came from experiments searching Preliminary Southern blot analysis using digested genomic DNA from hepatocytes and AS-30D hepatoma cells revealed that the HKII probe hybridized with much greater intensity to the hepatoma DNA than to the hepatocyte DNA. To estimate the differences in hybrid ization intensities we performed a dilution experiment. The hybrid ization signals with different amounts of EcoRI- and XbaI-digested AS-30D hepatoma genomic DNA were compared to the signal ob mined with 30 @gof DNA isolated from hepatocytes (Fig. 1). The for the HKH promoter region in these cells and in hepatocytes. Thus, six positive plaques were obtained when 5 X lO@plaques were screened from an AS-30D hepatoma genomic library, whereas only two positives were found in 2.5 X 106 plaques of a normal liver library. Taking into consideration that the liver library had been amplified previously, the estimated factor for amplification is near 6, in accordance with the results from Southern blot analysis. Instability of the genome is a well-known phenomenon of trans formed cells and amplification is a frequently observed mechanism for the overexpression of oncogenes, including N-myc (15) and the epidermal growth factor receptor gene (16). It is well known that a strong relationship exists frequently between a gene that is amplified blots were probed with two different probes specific for the HKIJ gene (Fig. 1, A and B). The intensities of the resulting bands indicate that 3—6 @ghepatoma DNA were equivalent to 30 ,.@ghepatocyte DNA. From this experiment, we estimated that AS-30D hepatoma cells contain approximately 5—10-foldmore copies of the HKII gene than normal hepatocytes. In addition, it is clear from Fig. lA that the signal intensities of all type II hexokinase-related bands obtained with AS and cell growth. The amplification of the HKII gene is consistent with 30D hepatoma DNA are the same. This indicates that the amplifica this relationship,becausethe role of this criticalmetabolicenzymeis tion extends to the whole coding region of the hexokinase gene. Moreover, when the membranes were probed again with DNA frag ments specific for the 5'-flanking region of the hexokinase gene (4), to provide cells with both energy and precursors for nucleotide and lipid biosynthesis. In a recent report (4), we provided evidence that increased expression of one or more transcription factors is involved similar results were obtained (data not shown). Thus, the amplified in the elevated production of HKH in AS-30D hepatoma cells. Work unit in AS-30D hepatoma cells also includes the promoter region of presented here suggests that amplification of the gene for the same the HKII gene. Densitometric quantification of autoradiograms made enzyme may play a role as well. Southern blot analysis (Fig. 1A) displayed some faint restriction A a b c d e f fragments with the hepatocyte DNA that were not observed in the AS-30D hepatoma DNA. As the restriction enzymes used, EcoRI and XbaI, are both sensitive to methylation of their recognition sequence, Cab this raisesthe possibilitythat methylationdifferencesexist withinthe kbp HKJI gene in normal hepatocytes and AS-30D hepatoma cells. Several studies reviewed in Ref. 17 have demonstrated that DNA methylation plays a role in gene regulation. Therefore, methylation could be 9.4 — 6.6 — involved in differential expression of HKII in normal and tumor cells. Additional experiments to test this hypothesis are in progress. For some oncogenes, it is well known that amplification is accom panied by recombination and rearrangement of the gene locus (18). To 4.4 — look for structural differences in the HKII gene locus in normal and AS-30D hepatoma cells, RFLP analysis was carried out. To circum vent problems due to methylation differences of normal and tumor DNA, methylation-insensitive restriction enzymes (RsaI, NdeI, HindllI) were used. For each enzyme, the same restriction fragment 2.3 2.0 — pattern is observed in both hepatocyte and AS-30D hepatoma DNA (Fig. 2). Thus, no macroscopic rearrangement of the hexokinase gene is seen at this level of resolution. Also, this result renders it unlikely that a translocation of the hexokinase gene locus has occurred in AS-30D hepatoma cells. Therefore, the amplification described above appearsto occurat the siteof theresidentgene,andthe possibilitythat S. B the HKII gene in AS-30D hepatoma cells has come under the control of different regulatory sequences through translocation seems remote. To obtain additional support for the amplification and localization of the HKJJ gene, in situ hybridization experiments were performed. Because primary hepatocytes divide very rarely and dedifferentiate rapidly, we used clone 9 (CRL 1439), a nontumorigenic, normal liver 9.4 6.6 Fig. 1. Amplificationof the HKIIgenomic sequencein AS-30D hepatomacells. High molecular weight genomic DNA was digested with EcoPJ and XbaI. Hepatocyte DNA, 30 @ag (Lane a); AS-30D hepatoma DNA, 30 ,sg (Lane b); and serial dilutions of the AS-30D hepatoma DNA (Lanes c—f, containing, respectively, 12, 6, 3, and 1.5 gagof DNA) were sizefractionatedon a 1%agarosegelandtransferredto a nylonmembrane.Theblotwas hybridizedto a HKHfull-lengtheDNA (A) or to a 26O-bpfragmentcorrespondingto the position — 197 to +63 of HKH(B). The blot was strippedof signal between hybridiza tions. Molecular weight markers(A-Hindffl) are shown to the left. Equal loading of hepatocyteand undilutedAS-30D hepatomaDNA was estimatedby ethidiumbromide staining of the gel (C). cell line (19) as a control for in situ hybridization. As shown in Table 1, these cells exhibit no detectable hexokinase activity, in contrast to AS-30D hepatoma cells where the activity is 762 mU/mg. The liver homogenate, which in addition to hepatocytes contains other cell types, exhibits a low but detectable hexokinase activity. In situ hy bridizations (Fig. 3) using the HKII cDNA as probe revealed that in AS-30D hepatoma cells, a signal could be detected readily in every (20/20)metaphase andinterphase cell.Occasional (4/20)tetraploid cells that were observed in the AS-30D hepatoma cell population showed a hybridization signal on two chromosomes, indicating that 2469 Downloaded from cancerres.aacrjournals.org on May 1, 2016. © 1996 American Association for Cancer Research. GENE AMPLIFICATION OF TYPE II HEXOIUNASE IN TUMOR CELLS abc d f e kbp abundant in AS-30D hepatoma cells than in control cells. Although FISH does notallow exact quantitation oftheamplification, itis consistent with at least a 5-fold increase in copy number. Moreover, the amplified sequence was localized to a single chromosome in AS-30Dhepatoma cells,suggesting thattheamplificationis present on only one of the two homologous chromosomes, a finding not uncommon for amplified genes (20). Chromosomally localized gene amplification represents one of the more stable fonns of amplified genes. Stable retention of amplified genes and their passage to daugh tar progeny are ensured only when such genes are integrated within a chromosome. Unstable amplified genes that are very common in transformed cells are associated characteristically with extrachromo somal elements called double minutes. However, double minutes were never observed in our studies of AS-30 hepatoma cells. In summary, results reported here provide for the first time cvi 9.4-6.6-4.4-- @11 — I,. A. 2.3 -. 2.0-.4 @ t 1I@!L@c:@ @ @ f . \ Fig. 2. RFLPanalysisofliver hepatocyteandAS-30D hepatomaDNA. GenomicDNA isolated from AS-SOD cells [10 ,.@g](Lanes a, c, and e) and hepatocytes [30 sag] (Lanes b, d@andI) was digestedto completion with methylation-insensitiverestrictionenzymes (Lanes a and b, RsaI; Lanes c and d@NdeI; Lanes e and f Hincffl), separated by gel electrophoresis, and transferred to a Hybond filter. The blot was hybridized to a fluores cein-labeled full-length HKII cDNA and developed by using an antifluorescein alkaline phosphatase conjugate and chemiluminescence. Hindffl-digested A-DNA was used as a marker. No differences between normal and tumor DNA were detected. Table 1 Hexokinase activity in normal AS-30Dhepatoma rat liver, hepatocytes (clone 9), and cellsHexokinase B. “Materialsand activity in whole-cell lysates was determined as described in Methods.― SD.Cell Values representthe mean of multipledatenninations± (mU/mg)Normalratliver source 1.2Hepatocytes 9)AS-30D (clone 52a hepatoma @ Hexokinaseactivity 12± 762 ± not detectable. the gene was amplified before the chromosomes were duplicated. The single positive chromosome seen in the AS-30D sample most likely represents the amplification site on one chromosome homologue only, but the loss of the other homologous chromosome cannot be ruled out. In contrast, in clone 9, no interphase signals were seen, and only 1 of 20 metaphase cells showed a faint specific signal. Because the probe used for in situ experiments was rather small (3.6 kb), genes with a low copy number cannot be detected easily with this size probe. This confirms again that the copy number of the HKtI gene is much more Fig. 3. in situ hybridization.The biotin-labeledprobe (pUC18, containingthe 111(11 cDNA) was hybridizedto metaphaseand interphasechromosomesfollowed by fluores cein immunodetection.A single block offluorescent signal was detectedeasily on a single chromosome of AS-30D hepatoma cells (A), whereas no signal was observed on the hepatocyte (clone 9) chromosomes (B). 2470 Downloaded from cancerres.aacrjournals.org on May 1, 2016. © 1996 American Association for Cancer Research. GENE AMPLIFICATIONOF TYPE IIHEXOKINASE IN TUMOR CELLS dence that a hexokinase gene (type II) is amplified in a tumor cell line exhibiting a high glucose catabolic phenotype. This amplification is stable, not associated with a rearrangement of the hexokinase gene locus, and occurs probably at the site of the resident gene. 10. Nakashima, R., Paggi, M. G., Scott, L. J., and Pedersen, P. L. 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In: Gene Amplification, pp. 317—333.Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. 2471 Downloaded from cancerres.aacrjournals.org on May 1, 2016. © 1996 American Association for Cancer Research. Glucose Catabolism in Cancer Cells: Amplification of the Gene Encoding Type II Hexokinase Annette Rempel, Saroj P. Mathupala, Constance A. Griffin, et al. Cancer Res 1996;56:2468-2471. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/11/2468 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. To request permission to re-use all or part of this article, contact the AACR Publications Department at permissions@aacr.org. Downloaded from cancerres.aacrjournals.org on May 1, 2016. © 1996 American Association for Cancer Research.